In PTL 1, a device for analyzing a biopolymer by irradiating the biopolymer passing through a nanopore with light to generate Raman scattering light, and measuring the light is described.
For the analysis of the Raman scattering light, a microspectroscopy device (a combination of a microscope and a spectroscopic optical system) called a Raman microscope is used, as described in PTL 2. In a Raman microscope, in order to observe weak Raman scattering light with high sensitivity and high resolution, an objective lens with high numerical aperture of nearly one or more is often used. In a microscope with such a high numerical aperture, the depth of field becomes submicron. Maintaining the distance between the sample and the objective lens stably for a long time at this level is difficult with only mechanical fixing, due to thermal expansion or the like. In the case of a single measurement such as a conventional microscope measurement, the focus can be adjusted for each measurement. However, at the time of long-term continuous measurement while a large biopolymer such as DNA is passing through a nanopore, performance of normal focusing becomes an interruption of the measurement and is not preferable. For such measurements, a real-time defocus correction (auto-focus) is preferred.
In conventional Raman microscopes, one excitation light beam is focused on one spot on a sample substrate by an objective lens to observe the light emission image on the spot. By observing the image on the spot, the occurrence of defocus can be detected but it is difficult to detect the direction.
Therefore, in a normal focusing, while measuring the image, shifting of the distance between the sample and the objective lens back and forth around the present position (acquisition of through-focus image) is necessary. This can be time consuming and is not suitable for real-time auto focus in continuous measurement.